Immunosuppressive action of 4-amino tetrahydrobiopterin

Tetrahydrobiopterin is a low molecular weight compound synthesized from guanosine 5` triphosphate, which is essential for specific hydroxylases, including the hydroxylation of aromatic amino acids and the biosynthesis of nitric oxide from L-arginine by nitric oxide synthases. In our preceeding project, we characterized the biochemistry of novel tetrahydrobiopterin derivatives, which had been developed in our laboratory. We found that these novel compounds were not only excellent tools to characterize the biochemical role of tetrahydrobiopterin in the nitric oxide synthase reaction, but also displayed exciting pharmacological properties. When applied to mice, the 4-amino analogue of tetrahydrobiopterin was at least as efficient as cyclosporin A in preventing allograft rejection. While cyclosporin A inhibited expression of interferon-gamma and of genes induced by this cytokine, the activation of the interferon-gamma axis in 4-amino tetrahydrobiopterin-treated animals remained as high as in control animals. Preliminary experiments suggest that the immunosuppressive potential of 4-amino tetrahydrobiopterin exceeds that of other nitric oxide synthase inhibitors. The aim of the present project is to unravel the molecular mechanisms of the immuno-suppressive action of 4- amino tetrahydrobiopterin.To achieve this, we plan to study the effects of 4-amino tetrahydrobiopterin on gene expression in cultured cells and cell lines, in mixed lymphocyte reactions, which constitute an in vitro model for immune reactions, and in tissues of hearts transplanted to allogeneic mice. These effects of 4-amino tetrahydrobiopterin will be studied in relation to other nitric oxide synthase inhibitors, to the tetrahydrobiopterin parent compound and to cyclosporin A, an immunosuppressive drug widely used in clinical applications.We want to first use the powerful expression array screening technique techniques, and then verify the observations by quantifying the corresponding protein levels in cells and tissue sections. We will then compare these results with results obtained using cells and tissues from mice lacking inducible nitric oxide synthase to dissect action dependent and independent from this enzyme. Finally, we plan to pinpoint the detailed mechanism by unravelling the signal transduction pathways responsible for the observed alterations in the expression profiles. With this project, we hope to characterize novel mechanisms of control of the immune response by pteridines. This knowledge could not only provide a step to the development of a new class of pterin-based immunosuppressive drugs, but also has the potential to identify novel targets for intervention in the immune system.

Our research focusses on the metabolic roles of pteridines. This class of substances comprises important vitamins such as folic acid and riboflavin, which are formed by bacteria and plants. Mammals have, however, the capacity to synthesize another pteridine, namely tetrahydrobiopterin. This compound is used as cofactor for specific hydroxylation reactions in our body, which are required for the biosynthesis of neurotransmitters, metabolism of essential amino acids and ether lipids, and the biosynthesis of nitric oxide, which in turn is important for neurotransmission, for the regulation of blood pressure and the defence against pathogens and tumours. In the project 16188 we investigated pharmacological actions of derivatives of tetrahydrobiopterin, which we had initially developed as tools to investigate the biochemistry of stimulation of nitric oxide synthases by tetrahydrobiopterin. One of these tetrahydrobiopterin analogues, 4-amino tetrahydrobiopterin, is an inhibitor of nitric oxide synthases and shows immunosuppressive action in animal models of septic shock and of allograft rejection. Our experimental approach was to study the action of tetrahydrobiopterin compounds on cultivated cells. We chose cells that constitute the immune systems host defence line. In a murine macrophage like cell line, we could demonstrate that tetrahydropterins down regulate gene expression and trigger programmed cells death. We succeeded in characterising the biochemical events leading to these effects: Tetrahydropterins in the medium convert oxygen to hydrogen peroxide. This triggers the activation of caspase 3, which in turn triggers programmed cell death and cleaves the P65 protein of the NF-kappa B transcription factor, which in turn causes the shutdown of gene expression. We then studied T-cells and dendritic cells, both key cellular constituents of the immune system that cooperate in the regulation of the immune response. We isolated these cells from mice with and without a functional gene for inducible nitric oxide synthase. In contrast to the macrophage cell line, these cells are resistant to the actions of hydrogen peroxide. We found that 4-amino tetrahydrobiopterin specifically attenuated the expression of a protein on the surface of dendritic cells, that is essential for its interaction with T-cells. This effect occurred irrespective of the presence or absence of a functional gene for inducible nitric oxide synthase. This novel immunosuppressive mechanism discovered in the project 16188 could possibly explain the immunosuppressive action of 4-amino tetrahydrobiopterin in animals, and may lead to the development of a new class of immunosuppressants.